Identification of residues crucially involved in soluble guanylate cyclase activation

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• 作者：Christiane Rothkegel ; Peter M. Schmidt ; Friederike Stoll ; Henning Schrö ; der ; Harald H.H.W. Schmidt and Johannes-Peter Stasch
• 关键词：Soluble guanylyl cyclase ; Nitric oxide ; Bay 41-2272 ; BAY 58-2667 ; Cyclic GMP
• 刊名：FEBS Letters
• 出版年：2006
• 出版时间：24 July 2006
• 年：2006
• 卷：580
• 期：17
• 页码：4205-4213
• 全文大小：606 K

文摘

The ubiquitous heterodimeric nitric oxide (NO) receptor soluble guanylate cyclase (sGC) plays a key role in various signal transduction pathways. Binding of NO takes place at the prosthetic heme moiety at the N-terminus of the β₁-subunit of sGC. The induced structural changes lead to an activation of the catalytic C-terminal domain of the enzyme and to an increased conversion of GTP into the second messenger cyclic GMP (cGMP). In the present work we selected and substituted different residues of the sGC heme-binding pocket based on a sGC homology model. The generated sGC variants were tested in a cGMP reporter cell for their effect on the enzyme activation by heme-dependent (NO, BAY 41-2272) stimulators and heme-independent (BAY 58-2667) activators. The use of these experimental tools allows the enzyme’s heme content to be explored in a non-invasive manner. Asp₄₄, Asp₄₅ and Phe₇₄ of the β₁-subunit were identified as being crucially important for functional enzyme activation. β₁Asp₄₅ may serve as a switch between different conformational states of sGC and point to a possible mechanism of action of the heme dependent sGC stimulator BAY 41-2272. Furthermore, our data shows that the activation profile of β₁IIe₁₄₅ Tyr is unchanged compared to the native enzyme, suggesting that Tyr₁₄₅ does not confer the ability to distinguish between NO and O₂. In summary, the present work further elucidated intramolecular mechanisms underlying the NO- and BAY 41-2272-mediated sGC activation and raises questions regarding the postulated role of Tyr₁₄₅ for ligand discrimination.